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Osteosarcoma (OS) is the most common bone tumor in children. Even though OS can affect patients at any age, the highest incidence occurs in adolescents. OS is a very aggressive and metastatic malignancy. Almost all patients have at least microscopic metastases at the time of diagnosis. When the patients with no clinically detectable metastasis were treated with surgery alone, only 11% of them had long-term disease free survival. The addition of chemotherapy improved the disease free survival rate to 69%. Patients with clinical metastasis at diagnosis have a worse prognosis. Death from OS is generally the result of progressive pulmonary metastasis with respiratory failure. Therefore, we need to identify the molecular mechanisms resulting in OS metastasis; mechanisms that could be targeted to create specific therapeutic agents. Ezrin is an intracellular protein that is involved in cell adhesion to extracellular matrix, cell-cell interactions, and signal transduction through growth factor receptors and Rho GTPases. In addition to these physiological functions, ezrin was also identified as a metastasis related gene in pediatric tumors. Both osteosarcoma and rhabdomyosarcoma showed increased expression of ezrin in highly metastatic tumors compared to less metastatic ones. Dr. Chand Khanna, our collaborator and a co-investigator in this grant, identified ezrin as a metastasis specific gene in a murine OS model. K7M2 was derived from K12 cells following three cycles of orthotopic tumor implant and pulmonary metastasis recovery. We have data from these cell lines showing enhanced chemotaxis in our in vitro motility assays. Furthermore, K7M2 cells express higher levels of ezrin protein than K12 and reducing ezrin protein expression decreases metastatic potential of K7M2 cells. Therefore, ezrin is a validated molecular target for prevention of metastasis in OS. We hypothesized that peptide and/or small molecules that can directly bind and inhibit ezrin function may also inhibit OS metastatic potential. In order to identify novel proteins that functionally interact with ezrin we are screening a peptide phage display library using ezrin as the target. In this experiment we will use a 12 aa random phage display library to discover novel peptide sequences that specifically bind to ezrin. We will identify mammalian proteins that contain homologous sequences to these peptides. Proteins identified by this method will be tested for direct interaction with ezrin and its biological outcome. Furthermoe, We will screen small molecule libraries for direct binding to ezrin using surface plasmon resonance technology. Small molecule libraries available from NCI will be tested for direct binding to recombinant ezrin molecule in Biacore T-100 machine utilizing surface plasmon resonance technology. We will then test the effect of ezrin binding peptides and small molecules in biochemical and biological assays that are directly related to OS metastasis, such as inhibition of ezrin’s biochemical activity in the cell and modulation of cellular motility in chemotaxis assays. Metastatic OS is a very difficult clinical challenge that impacts patient mortality. Since ezrin was identified as a metastasis related gene in OS, targeting ezrin function may be anti-metastatic. Discovery of peptides and small molecules capable of directly binding to ezrin and inhibiting its cellular functions will therefore have the potential to be clinically relevant tools. These molecules can serve as lead compounds for further optimization towards novel therapies. Hence, successful completion of our work may significantly benefit OS patients. Prevention or treatment of metastatic cells in OS will significantly improve the overall survival rate.

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